Gram's stain

Gram staining is a method for staining samples of bacteria that differentiates between the two main types of bacterial cell wall.

It is named after the inventor, the Danish scientist Hans Christian Gram (1853-1928), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria.

Gram Staining a Step by Step Procedure

1. First, an inoculum is taken from a culture using an inoculation loop and put on a slide. If the culture is solid, it is diluted by adding a drop of water on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put way too much inoculum at this step.
2. Heat-fix the specimen by passing the slide through a bunsen flame a few times, without allowing the slide to become hot to the touch.
3. Add a basic dye to stain the sample. crystal violet or gentian violet are suitable. Allow to stain for 1 minute. The slide should look violet in colour.
4. Rinse off with water for a maximum of 5 seconds.
5. Add iodine solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
6. Rinse with water.
7. Apply 95% ethanol or a mixture of acetone and alcohol several times until no more colour appears to come from the sample.This leaves Gram-positive organisms stained purple and Gram-negative organisms unstained.
8. Rinse with water.
9. Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or microscope

Gram-positive organisms will appear blue-black or purple.

Gram-negative organisms will appear red.

organisms that cannot reliably be differentiated by this staining technique are said to be Gram-variable

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. They also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" (the human body recognises a foreign body by its antigens, if they are hidden it becomes harder for the body to detect that bacterium. Often the presence of a capsule will increase the virulence of a pathogen)The human body does not contain peptidoglycan and in fact produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to penicillin.